The general aim of this proposal is to study the regulation, by pharmacological and electrical stimuli, of intracellular free calcium (Cai++) levels; and, to investigate the role of [Cai++] in the regulation of contraction of mammalian vascular smooth musacle. Although we know that Cai++ is essential for the contraction of vascular smooth muscle, the precise role of this ion in the initiation of contraction, the maintenance of tone and the relaxation of arteries and veins has not been defined due to the lack of a suitable method for recording Cai++ levels in smooth muscle. Similarly, the actual effect of pahrmacological agents on [Cai++] has not been directly determined. I will use a newly developed technique for loading the calcium indicator aequorin into smooth muscle. This technique, for the first time, permits Cai++ levels to be directly recorded in actively contracting mammalian blood vessels. Electrical stimulation and pharmacological agents will be studied, alone and in combination, to determine how they alter Cai++ levels and also how the resulting changes in [Cai++] affect tension development and maintenace. The effect of changes in [Cai++] on tension will be further investigated by altering transmembrane Ca++ influx with Ca++ chelators, inophores and Ca++ channel blockers. Intracelluar electrical activity will be recorded to determine if there is any correlation between membrane electrical events and the [Cai++] signals. The information obtained in this study should allow us to take a considerable step forward in understanding the role of [Cai++] in the regulation of smooth muscle contraction. Eventually, this should help us to understand how vascular muscle can undergo tonic contraction at a low energy cost and may tell us something about what goes wrong in cases of clinical spasm. The initial studies will be conducted on large, easily handled blood vessels but the later studies will extend to microcirculatory vessels. These small vessels are the major determinants of vascular resistance and blood flow distribution and spasm of these vessels has been implicated in a wide range of vascular diseases.